Decalcification of bones



United States Patent 3,396,222 DECALCIFICATTGN 0F BONES Archie B. Blackburn, Lake Jackson, T ex., assignor to The Dow Chemical Company, Midland, Mich., a corporation of Delaware No Drawing. Filed Feb. 3, 1965, Ser. No. 430,179 5 Claims. (Cl. 424-3) This invention relates to a process for the decalcification of bones by treatment with certain lactones and the hydrolysis products of such lactones. More particularly, the present invention relates to the decalcification of bones and bone-like tissues by contacting the bones or tissues with lactones containing from 3 to 7 carbon atoms and their hydrolysis products.

For biological studies, bones may be decalcified with a wide variety of chemicals, such as nitric, hydrochloric or acetic acids. However, the use of these acids for decalcification is usually unsatisfactory when subsequent histochemical studies are to be made, because extensive destruction of the tissues may result. Various specialized alternative methods which have been used to decalcify bones include electrolytic decalcification and the use of citric acid to prepare bone specimens in Which the enzymes have been preserved. Organic chelating agents (ion exchange resins and the sodium salt of ethylenediamine tetraacetic acid, for example) have also been used to decalcify bones prior to slicing the bones to prepare the specimens for microscopic examination. Such treatments generally have the disadvantage of requiring excessive time periods using dilute solutions so that the decalcification does not damage the structure of the tissue. The time required to decalcify a bone specimen by many of these prior art methods is a distinct disadvantage in diagnostic work based on the microscopic examination of bone tissue.

It has now been found that lactones of from 3 to 7 carbon atoms and the hydrolysis products of such lactones may be used to effectively and quickly decalcify bones and bone-like tissues prior to the sectioning or slicing of such tissues for pathological, histological or other scientific examination.

The lactones which are used in the process of the invention may be characterized by the following structural formula (wherein each R is as previously defined) and n is not zero when X is an oxygen atom. The main hydrolysis products obtained from these lactones include lactone polymers and the corresponding hydroxy acids formed according to the following equilibrium reactions:

0 0:0 H o R R o l r\ I u R-C- -o-n Ho-( )X(I3-o-orr 1 1) R H2O \R/n R (wherein R, n and X are as previously defined). Typical examples of suitable alkyl R groups (which may be the "ice same or different) include methyl, ethyl, n-propyl, ipropyl, n-butyl, i-butyl, sec.-butyl and tert.-butyl groups. Lactones such as 3,5-dimethyl-2-para-dioxanone, S-ethyl- Z-para-dioxanone, ,B-propiolactone, -butyrolactone and E-valerolactone, as well as the hydrolysis products of such lactones (especially the corresponding hydroxy substituted acids) may be used as decalcifying agents according to the process of the invention. As used herein, the term decalcification means that suflicient calcium has been removed from the specimen to provide a decalcified tissue which may be conveniently cut or sliced in relatively thin sections for microscopic, biological or pathological examination.

Typical hydrolysis equilibrium mixtures are represented by the following equations:

Lactones with rings containing from 4 to 6 atoms (and their hydrolysis products) form a preferred subclass of decalcifying agents.

The concentration of the lactone (or the equivalent amount of hydroxy acid theoretically obtainable by hydrolysis of this lactone to the acid form) in the decalcifying mixture is from about 1 mole to 10 moles of lactone per liter of decalcifying mixture (preferably from about 1.25 moles per liter to about 4.5 moles per liter) when aqueous compositions are employed. Below and above these limits, decalcification occurs, but at a much slower rate. The lactone (and/or their corresponding hydrolysis products) are most suitably employed in aqueous solutions. Other conventional fixative agents, preservatives, buffers and/or tissue staining materials may be incorporated into the lactone-containing bone decalcification composition. Aqueous or non-aqueous lactone solutions may be used. Water is preferred, but acetone, ethanol or isopropanol may also be employed as solvents. The decalcification of the bone specimens is generally complete in less than a two day period. A fixing agent such as formaldehyde, osmium tetroxide, alcohol or acetone may be used prior to or after treatment with the lactone (or lactone hydrolysis products). With the preferred concentrations and compositions of the invention, decalcification has been completed within a 6-8 hour period at room temperature to provide specimens which are easily sliced into very thin layers. Slicing may be accomplished with conventional apparatus such as a rotary or sliding microtome. Furthermore, the sliced sections do not show adverse effects when subsequently stained prior to a microscopic examination.

In a specific embodiment of the invention, a sample of bone from a specimen of the animal kingdom is immersed in an aqueous solution containing from 10 to percent by Weight of a lactone (such as 2-paradioxanone, beta-propiolacetone or the corresponding hydrolysis products: 2-hydroxyethoxyacetic acid and fi-hydroxypropionic acid) to extract sufficient calcium to render the bone sample soft enough to be sliced in thin sections without fracturing. The lactone may be in the form of an aqueous equilibrium mixture with its hydrolysis products. All percents by weight are based upon the non-hydrolyzed lactone for consistency in reporting results. The time required to decalcify a bone or bone-like tissue to 4. about to 50 percent by weight), immersion after one or two days did not produce any increase in the calcium concentration of the liquid extraction medium surrounding each bone sample. This indicates that the major an extent which will permit precision cutting may vary 5 proportion of the calcium is removed in the initial stages somewhat with the type of tissue, the size of the sample of the decalcification process. and the amount of calcium initially present. Time peri- Sections of all of the decalcified sheep bones (intact ods of 2 days or less are generally sufiicient for samples rib segments) revealed an accentually uniform histologic which are to be sliced for mounting and microscopic exappearance. The bony trabeculae stained brilliantly and amination. The decalcified sample is removed from the m normally. Cells within the bony lacunae were intact and aqueous lactone solution and then sliced, fixed, stained, showed the usual staining properties. Cells of the soft mounted or otherwise prepared for further histologic tissue of the bone marrow and adjacent periosteum were or histochemical evaluation. When Z-para-dioxanone is also intact and showed the usual staining properties when employed, it is not always necessary to use an addicompared to control bone material which was subjected tional fixative since the dioxanone (and its hydrolysis to nitric acid decalcificaton. products) possess preserving properties as disclosed in Iclaim as my invention: copending US. patent application 'Ser. No. 310,872, filed 1. A method of decalcifying a bone specimen which Sept. 23, 1963, now US. Patent No. 3,264,182. The eX- comprises: tent of the calcium removal may be followed chemi (a) contacting said bone specimen with an extracting cally (by analysis of the calcium concentration in the solution containing a compound of the formula lactone medium surrounding the bone sample) or physically (by a gross physical examination of the bone f r 0 0:0 properties of pliability, softness and translucency). I

The following examples are submitted for the purpose of illustration only and are not to be construed as limitl j ing the scope of the invention in any way. R R

Examples IX.General method wherein Bone samples (taken from the shattered femur of (1) of fromoto a freshly killed rabbit and segments of intact rib from (2) X sekced from the P' conslstmg of sheep) were Weighed and a 300 milligram sample of thfi oxygen atom and the dlvalent group rabbit bone tissue was immersed in 50 grams of treating solution. Bone samples of sheep rib were treated R by immersing 1.3 grams of the rib in 200 grams of treat- 1 ing solution. The treating solutions were produced by adding varying amounts of lactones to Water. The solu- R tions therefore contained an equilibrium mixture of the lactone and its hydrolysis products. The amount of caleach R PP 0f the formula t s zk'i cium in the solution surrounding the bone specimen was 111 k 15 an Integer of from and periodically determined to measure the increase in con- 40 n 18 g than Zero when X S Y centration as the decalcification proceeded. The calcium and the hydrolysis Products thereof, t0 form Soluble was determined using a Beckman Mode1 DU flame speccium salts by extraction of the calcium within the bone trophotometer. The Ca' absorption was measured at Specimen, and 422 m All of the bone samples which were treated were Separating thfi decalcified bone specimen of satisfactory for histologic evaluation. Excellent nuclear from the solubilized Calcium a detail is maintained when the decalcified samples are 2. The method of claim 1 wherein the concentrati n stained. The results are summarized in Table l. of said compound is from about 10 percent by weight TABLE 1 Concentration Calcium in Solution (Grams Per of Treating 100 Grams of Solution) After- Example Number Treating Compound Compound Type of Bone Tissue (Grams per lDay 2Days 5Days SDays 100 Grams of Solution) 0 1o 25 o 3,5-dimethyl-2-para-dioxanone- 25 2-hydroxyethoxyacetic acid 1 a 25 to about 50 percent by weight based upon the total weight of the extracting solution.

3. The method of claim 2 wherein the extracting solution contains water.

4. A method of preparing a bone specimen for pre- All of the bone samples which were treated with lactone (or the corresponding hydroxy acids) at a concentration of 10 to 50 percent by weight were flexible within 24 hours and could be cut with, a disecti ng knife or a microtome. In most cases (using concentrations of from 75 cision slicing which comprises contacting said bone specimen with an aqueous decalcifying solution containing from about 10 percent to about 50 percent by weight of 2-para-dioxanone in equilibrium with its hydrolysis products and then removing the decalcified bone specimen from the aqueous decalcifying solution.

5. A method of placing a fresh bone specimen in condition for precision slicing which comprises contacting said bone specimen with an aqueous decalcifying solution containing from about 10 percent to about 50 percent by weight of fi-propiolactone in equilibrium with its hydrolysis products and then removing the decalcified bone specimen from the aqueous decalcifying solution.

References Cited UNITED STATES PATENTS 5/1939 Lyons 167--65 OTHER REFERENCES Lacroix, Canadian J. Med. Tech., vol. 22, September 1960, pp. 78-82, 85.

Moodycliffe, Canadian J. Med. Tech., vol. 22, September 1960, pp. 87-91.

ALBERT T. MEYERS, Primary Examiner.

A. FAGELSON, Assistant Examiner. 

1. A METHOD OF DECALCIFYING A BONE SPECIMEN WHICH COMPRISES: (A) CONTACTING SAID BONE SPECIMEN WITH AN EXTRACTING SOLUTION CONTAINING A COMPOUND OF THE FORMULA 